Dysregulated apoptotic mechanisms are central to the pathogenesis and maintenance of cancer, and are major barriers to effective treatment. The BCL2 protein family, comprised of both pro- and anti-apoptotic members, controls the activation of downstream caspases, which are the major effectors of apoptosis. Thus developing small molecule probes that selectively target each anti-apoptotic protein family member may have relevance for both basic research and future clinical applications. We propose to screen for inhibitors of the anti-apoptotic protein A1. We have discovered that A1 is essential for cell survival in distinct subsets of cancer, including diffuse large B-cell lymphoma and melanoma. To our knowledge no small molecule inhibitor of A1 has been developed. We propose to collaborate with the MLPCN to employ a novel, validated high-throughput cell- based screen to identify inhibitors of A1. This cell-based screen presents A1 in its native state and allows us to discover compounds that can potentially modulate A1 protein function through multiple modes of binding. These include interaction at the conserved hydrophobic BH3 binding groove and thereby disrupt the interaction between A1 and its BH3-only protein partners (see Figure 1 and Section B) and also at modulatory allosteric sites of the A1 protein not necessarily conserved among BCL2 family proteins. Thus compared to conventional in vitro biochemical assays focusing on identifying compounds that bind to the conserved hydrophobic BH3-binding groove of anti-apoptotic proteins, our unique assay may broaden the spectrum of hit selection, increase the specificity among BCL2 family proteins, and will identify such hits in a physiologically relevant context. Following the primary screen, we will perform several validated secondary assays to identify compounds that act on A1-specific mechanisms. Data from those assays will be used to chemically optimize the hit compounds. These A1 inhibitors will serve as tools to further dissect the role of A1 in cancer. We anticipate that the proposed MLPCN collaboration will identify anti-A1 probe compounds that will greatly accelerate the study of apoptosis mechanisms in cancer. PUBLIC HEALTH RELEVANCE: Dysregulated apoptotic mechanisms are central to the pathogenesis and maintenance of cancer, and are major barriers to effective treatment. We have discovered that the anti-apoptotic protein A1 is essential for cell survival in distinct subsets of cancer, including diffuse large B-cell lymphoma and melanoma, yet no small molecule inhibitor of A1 has been developed. We propose to collaborate with the MLPCN to employ a novel, validated high-throughput cell-based screen to identify inhibitors of A1.